Project Summary/Abstract The medial prefrontal cortex (mPFC) is one of the key brain regions implicated in reinstatement of ethanol-seeking behaviors. Recent findings from our laboratory demonstrates that the mPFC harbors progenitors (progeny of stem cells that are characterized by limited self-renewal) that proliferate, mature and differentiate into premyelinating and myelinating oligodendrocyte progenitor cells (OPCs), and generate myelinating oligodendroglia. Most notable is that increased ethanol self-administration during chronic intermittent ethanol vapor inhalation (drinking during dependence; CIE) reduce newly born OPCs during ethanol experience. However, withdrawal and forced abstinence from CIE produces a rebound effect in the proliferation and survival of newly born mPFC OPCs, visualized as increases in the number of premyelinating OPCs and myelinating oligodendroglia and these changes are associated with increased expression of myelin associated proteins. These alterations in CIE rats also predicted enhanced ethanol seeking behaviors, indicating that dependence-like behavior in CIE animals produced profound alterations in the cellular composition of mPFC which could be a risk factor for enhanced propensity for relapse. We also demonstrate that increased generation of newly born OPCs during forced abstinence in CIE rats positively correlate with enhanced expression of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD-31; a marker of neuroinflammatory response) in the mPFC and enhanced propensity for reinstatement of ethanol seeking. Furthermore, preliminary whole-cell patch-clamp data from electrophysiology studies performed during forced abstinence in CIE rats demonstrate that enhanced generation of mPFC OPCs, and expression of myelin associated proteins and PECAM-1 is associated with enhanced basal synaptic transmission and neuronal excitability of layer 2/3 mPFC pyramidal neurons indicating hyperexcitability and enhanced glutamatergic transmission in the mPFC. Based on our published and pilot data we hope to mechanistically link enhanced generation of OPCs and expression of PECAM-1 in the mPFC in promoting hyperexcitability of glutamatergic neurons and enhanced relapse to ethanol seeking. We propose that inhibiting the aberrant generation of newly born OPCs, myelin associated proteins and PECAM-1 response during forced abstinence will preserve neuronal function within the mPFC and thus reduce reinstatement of ethanol seeking. Our studies combining robust behavioral models of alcohol addiction and relapse, cellular and biochemical alterations in the mPFC in combination with functional electrophysiological studies offer a powerful mechanistic context to better understand the factors associated with enhanced propensity for relapse.